The long term goal of this competing renewal is the development of methods that allow for the analysis of various enzymatic processes using mass spectrometry. The major areas proposed herein include: 1) Kinetics and catalytic mechanism determination for phosphohexose-isomerases. 2) Kinetics, substrate determination, and mechanism determination of tyrosylprotein sulfotransferases. This will also include a new procedure for unambiguous determination of sites of sulfation on natural substrates. 3) Investigation of substrate and inhibitor interactions with a newly discovered human endosulfatase, Hsulf-2 Each of these areas employs electrospray ionization (ESI)mass spectrometry (MS)using either an LTQ ion trap or an FTICR instrument to measure various kinetic parameters such as Km, Vmax, Ki, kcat and specificity constants. It is proposed that kinetic parameters can be calculated using ESI-MS thus obviating the need for chromophores as required by traditional spectrophotometric techniques. Equally important is the fact that kinetic information, either previously intractable or difficult to measure, can be gleaned using the MS based methods outlined herein. The problem with analyzing sulfated biomolecules is well recognized in mass spectrometry. The proposed research will alleviate those problems associated with loss of sulfate upon electrospray ionization and MS/MS in the positive ion mode. Similarly, methods are proposedfor obtaining biological data on phosphoisomerase systems; one of which is highly difficult to classify kinetically and mechanistically. . .